Hemogenic endothelial cells (HE) represent a rare cell population that gives rise to hematopoietic stem cells (HSCs) through a process referred to as endothelial-to-hematopoietic transition (EHT). EHT is a key developmental stage required to produce the mature, adult hematopoietic system. The underlying mechanisms that regulate human definitive hematopoiesis remain poorly elucidated. Human embryonic stem cells (hESCs) provide a well-defined cellular platform that can be used to study these mechanisms and to improve the in vitro production of HSCs potentially suitable for clinical applications. Recent studies suggest the aryl hydrocarbon receptor (AHR) is a critical regulator of HSC development. However, it is unknown whether AHR signaling is required for definitive hematopoietic induction from human HE. My preliminary studies suggest that AHR is expressed in low levels in undifferentiated hESCs and becomes upregulated immediately after hematopoietic induction. Based on these studies in hESCs and other developmental models, I hypothesize AHR negatively regulates early hematoendothelial development from human HE. I will test this central hypothesis in two specific aims. In Specific Aim 1, I will determine the rol of AHR in definitive hematopoietic development from hESCs. I hypothesize blocking or eliminating AHR signaling will enhance definitive hematopoietic development. Our lab has formuated well-defined conditions to support the differentiation of hematopoietic cells from hESCs. Here, I will utilize small molecule antagonists and agonists of the AHR as well as CRISPR/Cas9 endonuclease gene editing to assess the effects AHR has in generating definitive hematopoietic cells. In Specific Aim 2, I will perform single-cell genomic analysis to characterize human HE and non-HE subpopulations derived from hESCs. Previous studies are confounding since there is no universally accepted definition of HE and most studies examine a heterogenous population of cells. My preliminary studies have utilized a combination of genetic expression with RUNX1c, a key EHT gene, with a CD34+CD144+CD41-CD43- phenotype that allows us to isolate a putative HE population derived from hESCs. Using these cells, I hypothesize a distinct HE population with a unique genetic signature can be identified in cells derived from hESCs. This signature will further allow us to determine how key hematopoietic and endothelial genes are regulated by the AHR signaling pathway. Collectively, these studies are significant in that they will provide novel information on the identity of human HE and how the AHR pathway contributes to EHT. The results of these studies will be broadly applicable in improving the production of patient-specific HSCs suitable for transplantation in individuals with various hematological diseases. Moreover, this application provides a rigorous, yet defined scientific and mentoring framework to foster my career goals of becoming a successful academic physician-scientist.